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Am J Physiol Gastrointest Liver Physiol 292: G53-G65, 2007. First published August 17, 2006; doi:10.1152/ajpgi.00118.2006
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HORMONES AND SIGNALING

ApoB mRNA editing is mediated by a coordinated modulation of multiple apoB mRNA editing enzyme components

Zhigang Chen,1 Thomas L. Eggerman,2,3 and Amy P. Patterson1,4

1National Heart, Lung and Blood Institute; 2Division of Diabetes, Endocrinology, and Metabolic Diseases, National Institute of Diabetes, Digestive, and Kidney Diseases; 3Department of Laboratory Medicine, Warren Grant Magnuson Clinical Center; and 4Office of Biotechnology Activities, National Institutes of Health, Bethesda, Maryland

Submitted 15 March 2006 ; accepted in final form 15 August 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Apolipoprotein (apo)B mRNA editing is accomplished by a large multiprotein complex. How these proteins interact to achieve the precise single-nucleotide change induced by this complex remains unclear. We investigated the relationship between altered apoB mRNA editing and changes in editing enzyme components to evaluate their roles in editing regulation. In the mouse fetal small intestine, we found that the dramatic developmental upregulation of apoB mRNA editing from ~3% to 88% begins with decreased levels of inhibitory CUG binding protein 2 (CUGBP2) expression followed by increased levels of apoB mRNA editing enzyme (apobec)-1 and apobec-1 complementation factor (ACF) (4- and 8-fold) and then by decreased levels of the inhibitory components glycine-arginine-tyrosine-rich RNA binding protein (GRY-RBP) and heterogeneous nuclear ribonucleoprotein (hnRNP)-C1 (75% and 56%). In contrast, the expression of KH-type splicing regulatory protein (KSRP), apobec-1 binding protein (ABBP)1, ABBP2, and Bcl-2-associated athanogene 4 (BAG4) were unaltered. In the human intestinal cell line Caco-2, the increase of apoB mRNA editing from ~1.7% to ~23% was associated with 6- and 3.2-fold increases of apobec-1 and CUGBP2, respectively. In the mouse large intestine, the editing was 48% and had a 2.7-fold relatively greater CUGBP2 level. Caco-2 and the large intestine thus have increased instead of decreased CUGBP2 and a lower level of editing, suggesting that inhibitory CUGBP2 may play a critical role in the magnitude of editing regulation. Short interfering RNA-mediated gene-specific knockdown of CUGBP2, GRY-RBP, and hnRNP-C1 resulted in increased editing in Caco-2 cells, consistent with their known inhibitory function. These data suggest that a coordinated expression of editing components determines the magnitude and specificity of apoB mRNA editing.

apolipoprotein; development; apolipoprotein B mRNA editing enzyme; apolipoprotein B mRNA editing enzyme-1 complementation factor; CUG binding protein-2

APOLIPOPROTEIN B (apoB) plays a central role in lipoprotein metabolism (40). ApoB protein exists mainly in two isoforms: apoB-48 and apoB-100. Only synthesized by the intestine in the human, apoB-48 is essential for the assembly of chylomicrons and has an obligatory role in the intestinal absorption of dietary fats. ApoB-100, which is produced in the liver in humans, is required for the synthesis and secretion of VLDL (40). ApoB-100 is virtually the only protein component of LDLs, which are metabolic products of VLDL and contain about two-thirds of the cholesterol in human plasma. Elevated concentrations of apoB-100 and LDL-cholesterol in plasma are recognized risk factors for developing atherosclerotic coronary artery disease (40).

ApoB-48 is generated by the editing of apoB mRNA. The edited apoB RNA specifies an UAA translational stop codon at position 2153 and encodes a truncated product referred to as apoB-48. ApoB-48 is a peptide 48% the length of apoB-100 and is collinear with the amino-terminus of apoB-100 (2, 40). As a major transport protein of cholesterol in plasma, apoB-100 is responsible not only for the solubilization of lipid but also for its target delivery to cells throughout the body. This latter function is accomplished through the interaction of the apoB-100 carboxyl-terminus with a cell surface receptor, the LDL receptor (LDLR). ApoB mRNA editing results in a protein missing in the carboxyl-terminus and has no intrinsic capacity to interact with the LDLR (1). Dietary lipids are packaged into lipoprotein particles called chylomicrons, which contain apoB-48. These particles are cleared by a distinct receptor pathway that recognizes another ligand on the surface of the particle, namely, apoE. Thus, apoB mRNA editing provides a dual ligand-receptor pathway for the distribution of cholesterol and other lipids throughout the body (1), playing an important physiological role in mammalian lipid metabolism.

ApoB mRNA editing is an enzymatic modification of a single cytidine in a large transcript with >14,000 residues. A minimal requisite sequence region size of ~30 nucleotides flanking the editing base has been identified. A conserved motif is comprised of 11 nucleotides, located 4–6 nucleotides downstream of the edited base, and has been shown to be particularly critical for in vitro RNA editing. Virtually all mutations in this region eliminate the C to U deamination of a synthetic template (5, 33). The assembly of a sequence-specific higher-order "editosome" complex on the RNA-specific region mediates apoB mRNA editing. The size of the editosome was identified to be 27S in sucrose gradients using complexes assembled in vitro (34, 36). The minimal enzyme complex that can mediate C to U editing of apoB RNA in vitro comprises two proteins: apoB mRNA editing enzyme, catalytic polypeptide 1 (apobec-1), the catalytic subunit, and apobec-1 complementation factor (ACF), a novel RNA-binding protein that serves as the RNA recognition component of the editing enzyme (26, 28, 38). Other proteins including CUG binding protein 2 (CUGBP2), glycine-arginine-tyrosine-rich RNA binding protein (GRY-RBP), heterogenous nuclear ribonucleoprotein (hnRNP)-C1, apobec-1 binding protein (ABBP)1, ABBP2, KH-type splicing regulatory binding protein (KSRP), Bcl-2-associated anthogene 4 (BAG4), and auxiliary factor (AUX)240 have been identified by using detection assays that primarily reflect their ability to interact with apobec-1, ACF, or apoB RNA (3, 7, 19, 21–26, 32). However, the specific functional role that each candidate auxiliary protein might have in the editing complex remains to be clarified, and the precise composition of the holoenzyme in vivo is yet to be elucidated.

Although apoB mRNA editing is a precise and efficient deamination reaction of a single cytidine in the molecule under physiological conditions, overexpression of apobec-1 leads to unregulated and nonspecific mRNA editing, called hypermutation (37). How these enzyme components are regulated and assembled into the editing complex in vivo to accurately perform apoB mRNA editing remains unknown. ApoB mRNA editing is tissue specific, and the efficiency of editing is modulated metabolically, hormonally, and during development without hypermutation. Many factors have been reported to be able to regulate apoB mRNA editing in the rodent liver or hepatic cells. In addition to the developmental stage (15, 39), interventions such as growth hormones, estrogen, thyroid hormones, insulin, fasting, carbohydrate refeeding, treatment with ethanol, modulation of calcium concentration, and phosphorylation status affect editing (8, 9, 12, 14, 27, 30). However, the exact molecular mechanisms that regulate apoB mRNA editing activity remain largely unclear.

The small intestine plays a primary role in the adsorption and transport of dietary lipids and is the major organ for apoB mRNA editing under physiological conditions in humans, whereas in rodents the liver also edits apoB mRNA. The small intestine undergoes significant changes during fetal development. The intestinal endoderm undergoes cytodifferentiation to form the functional unit crypt-to-villus axis of the intestinal epithelium. This crypt-to-villus axis is a continuous and rapidly renewing system involving cell generation, migration, and differentiation from the stem cell population located at the bottom of crypt to the extrusion of the terminally differentiated cells at the tip of the villus (31a). During fetal development, apoB mRNA editing is increased from ~3% at day 15 to ~88% at day 18 postconception in the mouse small intestine (30). The developmental regulation of apoB mRNA editing is an autonomous function of the small intestine and is coincident with the onset of intestinal ontogenesis characterized with the formation and maturation of the crypt-to-villus axis (12, 30, 31, 39). The dramatic developmental upregulation in a short period in the fetus is not only a physiologically important event associated with lipid adsorption and transportation from the small intestine but also provides a good model to understand the regulation of the apoB mRNA editing complex.

To evaluate how the editing component candidates contribute in apoB mRNA editing regulation and assess their importance in the editing complex, we investigated their gene expression ontogenesis in the mouse small intestine, the mouse large intestine, the human intestinal cell line Caco-2, and mouse and rat liver cell lines BNL CL2 and FAO. We found that the dramatic developmental upregulation of apoB mRNA editing in the mouse small intestine begins with downregulation of inhibitory CUGBP2 expression followed by upregulation of apobec-1 and ACF and then by downregulation of the inhibitory components GRY-RBP and hnRNP-C1. The expression of other editing components is relatively unaltered. Short interfering (si)RNA-mediated gene-specific knockdown of CUGBP2, GRY-RBP, hnRNP-C1, KSRP, ABBP1, BAG4, and ABBP2 all resulted in an editing increase in Caco-2 cells, indicating that these components directly or indirectly play an inhibitory role in the editing process. The data demonstrate that the regulation of apoB mRNA editing is mediated by a coordinated modulation of multiple editing enzyme components.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal treatment. CD-1 strain mice (~8 wk old), obtained from Charles River Laboratories, were housed in metabolic cages and fed with regular chow. One male and one female mouse were housed in one cage overnight and separated the next day. The female mice for each group were then housed together. At various time points postconceptionally, the middle portion of the small intestine was removed from three to seven fetal or newborn mice from a single litter. Total RNA was extracted from the combined litter tissues using TRIzol reagent (Invitrogen) following the manufacturer's instructions. For each time point, two to four separate samples were prepared for analyses. For the comparison study between the small and large intestine, the tissues were taken from 8-wk-old normal adult female mice, and total RNA was extracted as above.

The protocol under which the animal work in this study was done was approved by an independent review committee at the Center for Biologics Evaluation and Research, a center within the Food and Drug Administration.

Cell culture. The human colon adenocarcinoma cell line Caco-2 and mouse and rat liver cell lines BNL CL2 and FAO were purchased from the American Type Culture Collection (Rockville, MD). Caco-2 and BNL CL2 cells were maintained in DMEM containing 10% FBS. FAO cells were maintained in RPMI-1640 containing 10% FBS. For the developmental analyses, Caco-2 cells were plated on either plastic 35-mm six-well dish plates or a membrane filter insert (Millicell-HA, Millipore). Approximately 1 x 106 cells were placed in each well, and cells reached confluency in 2 days. Culture media were changed every 2 days. Total RNA was extracted from the cultured cells at the time points indicated using TRIzol reagent (Invitrogen). For each time point, three separate samples were prepared for analyses.

ApoB mRNA editing analyses. The endogenous apoB mRNA editing in cells was determined by primer extension following RT-PCR amplification as previously described (30). Total RNA samples were pretreated with DNase RQ1 (Promega). First-strand cDNA was generated with a random primer using murine Moloney leukemia virus (M-MLV) reverse transcriptase (Invitrogen), and PCR was performed for the amplification of apoB. The PCR conditions were as follows: 2 min at 94°C; 35 cycles of 0.5 min at 94°C, 1 min at 55°C, and 2 min at 72°C; and 7 min at 72°C. The RT-PCR amplicon of apoB flanking the edited base were then purified using a GeneClean II kit (Bio 101) and annealed to an antisense 32P-labeled oligonucleotide (apoB extension primer) at 42°C for 1 h. Primer extension was performed at 42°C for 10 min as previously described (30). The primer extension products were then resolved on an 8% polyacrylamide-urea gel, and the ratio of edited to unedited apoB mRNA was determined by a PhosphoImager.

mRNA abundance quantitation by RT-PCR. Total RNA (5 µg) was pretreated with DNase RQ1 (Promega), and first-strand cDNA was generated with a random primer using M-MLV reverse transcriptase (Invitrogen). cDNA aliquots with the same volume were amplified by PCR for the selected genes in the presence of 0.4 µl [{alpha}-32P]dCTP (3,000 Ci/mmol, 10 Ci/ml; Amersham). PCR amplification was performed with selected cycles determined according to the gene abundance in a RoboCycler Gradient-40 (Stratagene) as follows: 2 min at 94°C; 21–40 cycles of 30 s at 94°C, 1 min at 55°C, and 2 min at 72°C; and 7 min at 72°C. The primers used for each gene analysis are listed in Table 1. The primer sets designed for mouse GRY-RBP, ABBP2, KSRP, and BAG4 were utilized for the PCR amplification of corresponding genes in rat samples because the gene sequences for those rat genes are not available. RT-PCR products of the corresponding size were identified and counted for gene abundance. Similarly, the human ABBP1 primer set was also used for the quantitation of mouse and rat ABBP1. The resultant PCR products (10-µl aliquot) were resolved by 6% PAGE and quantified by a PhosphoImager. The abundance of each gene was determined by the PCR product counts normalized to housekeeping gene beta-actin for mouse samples, GAPDH for Caco-2 cells, and beta2-microglobulin (beta2M) for rat samples and was graphically represented. The means and SDs were determined by the Microsoft Excel program, and the statistical multicomparison significance was determined by the StatView program (ANOVA/Fisher protected least-significant difference test and Schefle F-test).


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  		Table 1. Oligonucleotide primers used in this study

 
For the quantitation of alternative splicing isoforms of apobec-1/T, hnRNP-C1/2, and ABBP1/hnRNP-A/B1, one pair of primers covering the deletion region of the genes was selected, and the abundance for each gene was calculated based on the counts of each individual band and half of heteroduplex band as previous reported (10). For the comparison of the expression levels between the small and large intestine in GoGoGoGoFig. 5, RT-PCR was performed without labeling with [{alpha}-32P]dCTP and the abundance was determined by the density using a PhosphoImager after bands were separated on a 2% agarose gel.


Figure 1
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  		Fig. 1. Developmental regulation of apolipoprotein B (apoB) mRNA editing in the mouse small intestine is associated with altered expression of multiple editing enzyme components. Mouse small intestine tissues of different developmental ages were obtained, and cDNA was generated from the tissue total RNA extract by the random primer method. A: apoB mRNA editing analyses. An apoB fragment covering the editing site was PCR amplified from cDNA, and the editing levels were determined by primer extension followed by separation by 8% PAGE. B: mRNA expression analyses of multiple genes. cDNA aliquots underwent semiquantitative PCR amplification in the presence of [{alpha}-32P]dCTP, and the abundance of each gene was determined according to their PCR product counts after separation by 6% PAGE.

 

Figure 2
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  		Fig. 2. Graphical representation of the data shown in Fig. 1. The levels of apoB mRNA editing and mRNA expression of different genes were determined by a PhosphoImager and analyzed using the Microsoft Excel program. The gene expression levels represents levels after normalization to the housekeeping gene beta-actin. Each bar represents the mean and SD of the group with n = 2–4.

 

Figure 3
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  		Fig. 3. Regulation of apoB mRNA editing in Caco-2 cells cultured on filters. Caco-2 cells were cultured on millicell-HA filters and harvested at various time points. cDNA was generated from the total RNA extract by the random primer method. cDNA aliquots were analyzed for apoB mRNA editing or mRNA expression of different genes by PCR amplification in the presence of [{alpha}-32P]dCTP. The resultant PCR products were resolved by 6% PAGE, and the gene expression levels were determined by a PhosphoImager. Data are normalized to the housekeeping gene GAPDH, and each bar represents the mean and SD of the group with n = 3.

 

Figure 4
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  		Fig. 4. Comparison of apoB mRNA editing and CUGBP2 expression in Caco-2 cells cultured on plates. Caco-2 cells were cultured on plates and harvested at various time points. cDNA was generated from the total RNA extract by the random primer method, and cDNA aliquots were analyzed for apoB mRNA editing or mRNA expression of different genes by PCR amplification in the presence of [{alpha}-32P]dCTP as in Fig. 3. Data for CUGBP2 expression are normalized to the housekeeping gene GAPDH, and each bar represents the mean and SD of the group with n = 3.

 

Figure 5
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  		Fig. 5. Differential relative expression of CUGBP2 in the mouse small and large intestine. Samples from the small and large intestine were taken from 8-wk-old female mice, and total RNA was extracted. Aliquots of the cDNA generated by the random primer method were analyzed for apoB mRNA editing levels and gene abundance. A: apoB mRNA editing analyses. An apoB fragment covering the editing site was PCR amplified, and the editing levels were determined by primer extension followed by separation by 8% PAGE. B: mRNA expression level analyses of different genes. cDNA aliquots were PCR amplified, and the resultant PCR products were resolved using 2% agarose gel electrophoresis. C: graphic representation of the data in B after normalization to beta-actin. The abundance of each gene was determined according to their density counts by a PhosphoImager. Each bar represents the mean and SD with n = 3. *Statistically significant differences between groups (P < 0.01).

 
siRNA-mediated gene-specific knockdown in Caco-2 cells. siRNA against ACF, CUGBP2, GRY-RBP, hnRNP-C1, KSRP, ABBP1, BAG4, and ABBP2 were selected according to the predesigned collection by Ambion. Two or three predesigned siRNA for each gene were chemically synthesized and annealed for the gene knockdown tests in Caco-2 cells by transient transfection with siPORT NeoFX (Ambion) according to the manufacturer's instruction. The effective siRNAs for each gene used for the data presentation were listed according to their identification with Ambion as follows: ACF siRNA, 123860; CUGBP2, 135875; GRY-RBP, 135713; hnRNP-C1, 145409; KSRP, 121459; ABBP1, 145406; BAG4, 137797; and ABBP2, 134961. A scrambled siRNA was used as the negative control.

To evaluate the effect of siRNA-mediated gene knockdown of each gene on apoB mRNA editing in Caco-2 cells, 2.3 x 105 cells were plated together with siRNA-siPORT NeoFX complexes. Twenty-four hours after the transfection during the cell plating, apobec-1 was introduced into the siRNA-transfected Caco-2 cells by an infection with an adenovirus expressing apobec-1 (apobec-1-adenovirus; multiplicity of infection ~3). Forty-eight hours later, apobec-1-adenovirus was removed by replacing with normal growth media, and, 72 h later, cells were harvested for total RNA extraction by TRIzol reagent (Invitrogen). The levels of editing and gene expression for each treatment were determined using the total RNA extract.

For the preparation of apobec-1-adenovirus, apobec-1 from the human small intestine was PCR amplified and cloned into the vector pENTRA1 (Invitrogen) using BamHI and EcoRI sites and then transferred into the adenoviral expression vector pAd/CMV/V5 (Invitrogen) by recombination following the manufacturer's instructions. Apobec-1-adenovirus was generated by transfection of apobec-1-pAd/CMV/V5 linealized with PacI in 293A cells, and the virus collected from the media after cells had been lysed by freezing and thrown for three times was titrated according to the manufacturer's instruction.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Developmental regulation of apoB mRNA editing is associated with the coordinate modulation of multiple enzyme components. To evaluate the functional roles that each candidate apobec-1 auxiliary protein might have in regulating editing, a correlation between the levels of apoB mRNA editing and expression levels of the editing complex components during mouse intestine development was investigated. The mRNA expression of each editing enzyme component was determined by semiquantitative RT-PCR utilizing mouse small intestine tissues of different developmental ages ranging from 14 days postconception to 12 days after birth (postconception 31 days). As shown in Figs. 1A and 2A, apoB mRNA editing increased from 2.6% at postconceptional day 14 to an adult level of 91.5% at day 19. The most dramatic editing change occurred in a period of only 3 days (from days 15 to 18). Gene expression levels significantly changed for apobec-1, ACF, CUGBP2, GRY-RBP, and hnRNP-C1 over this 3-day period, whereas the housekeeping gene beta-actin remained constant, as shown in Fig. 1B. Apobec-1 is the catalytic component, and ACF is an essential activating cofactor of the editing enzyme complex. When apoB mRNA editing increased from day 15 (3.3%) to day 17 (71.3%), apobec-1 and ACF increased 4.4- and 8.6-fold, respectively, and remained at these levels thereafter (Figs. 1B and 2, B and C). A temporal correspondence between the altered expression of apobec-1 and ACF and the editing change was observed. This is consistent with the finding that apobec-1 and ACF comprise the minimal active requirement for in vitro apoB mRNA editing (26, 28, 38).

In contrast to the increasing levels of the active components apobec-1 and ACF, the expression of editing inhibitory components, CUGBP2, GRY-RBP, and hnRNP-C1, decreased significantly. As shown in Figs. 1B and 2, GRY-RBP and hnRNP C1 gradually decreased after day 16 and had up to 75% and 57% decreases by day 21 compared with day 14. The expression of CUGPB2 decreased 50% on day 15, 1 day ahead of the editing increase, and had an 89% decrease by day 18. These data demonstrate that CUGBP2 decreases significantly prior to editing increases, whereas both GRY-RBP and hnRNP-C1 decrease during and after the editing increase, indicating that CUGBP2 is a critical factor contributing to the dramatic increase in apoB mRNA editing. There were no significant and consistent changes observed for ABBP1, ABBP2, KSRP, and BAG4 (Figs. 1B and 2), indicating that they may not contribute directly to the upregulation of apoB mRNA editing. These data suggest that the dramatic increase of apoB mRNA editing results from an initial downregulation of CUGBP2 followed by upregulation of apobec-1 and ACF and downregulation of GRY-RBP and hnRNP-C1.

Developmental regulation of apoB mRNA editing in Caco-2 cells is also associated with mRNA expression modulation of multiple editing enzyme components. The human intestinally derived cell line Caco-2 develops the capacity to edit apoB mRNA as the cellular phenotype matures (15, 16). Caco-2 cells cultured on membrane filters or plastic plates offer in vitro cellular models mimicking intestinal development. As shown in Fig. 3, a pattern similar to the intestinal developmental regulation of apoB mRNA editing was observed with filter-grown Caco-2 cells. The editing increased from a basal level of 1.7% on day 4 to 23.4% on day 16, with the most significant changes occurring from day 6 (2.9%) to day 10 (18.1%). This significant editing upregulation was accompanied by significantly altered expression of apobec-1 and CUGBP2 and a modest decline of hnRNP-C1 (Figs. 3, B, F, and H). Increased apobec-1 correlated well with increased editing (Fig. 3, A and B), like the mouse small intestine, increasing 4.0-fold by day 10 relative to day 4, when editing increased 10-fold. The catalytic editing component apobec-1 has two RNA alternative splicing variants, named apobec-1 and apobec-T, in Caco-2 cells (20). More than 90% of apobec-1 is inactive apobec-T in Caco-2 cells. The expression pattern of apobec-T was identical with apobec-1 (data not shown). As shown in Fig. 3F, CUGBP2 increased 3.8-fold from day 4 to day 18, when editing increased from 1.7% to 23.3%. Unlike the mouse small intestine, inhibitory CUGBP2 expression increased in Caco-2 cells, suggesting that it may contribute to the lower level of Caco-2 editing. CUGBP2 expression increased significantly between days 4 and 6, demonstrating that CUGBP2 was modulated prior to the increase in editing, as was observed in the small intestine. The increased expression of inhibitory CUGBP2 occurring prior to the increase in editing indicates that it is an early response in the pathway regulating editing.

Like Caco-2 cells cultured on filters, Caco-2 cells cultured on plastic plates also offered a similar pattern of apoB mRNA editing changes, albeit with lower magnitude (Fig. 4A). CUGBP2 expression increased 3.2-fold from day 4 to day 6 and remained at the level thereafter, whereas the editing remained unchanged from day 4 to day 6, increased from 0.7% on day 6 to 1.4% on day 8, and then reached the highest point (2.0%) on day 12. These data confirmed that CUGBP2 was modulated prior to apoB mRNA editing in filter- and plastic-cultured Caco-2 cells, as was observed with the mouse small intestine.

CUGBP2 expression is increased significantly in association with decreased apoB mRNA editing in the mouse large intestine compared with the small intestine. The modulation of CUGBP2 in Caco-2 cells was opposite to that in the small intestine, which might explain the lower levels of apoB mRNA editing in Caco-2 cells. The colonic origin of Caco-2 cells led us to investigate if CUGBP2 was highly expressed in the large intestine, contributing to the decreased apoB mRNA editing observed in the colon compared with the small intestine. As shown in Fig. 5, apoB mRNA editing was 81–88% in the small intestine compared with 47–49% in the large intestine. The gene expression analyses demonstrated that the largest relative difference among the tested components occurred with CUGBP2, where the expression level was 2.7-fold relatively higher in the colon after being normalized to beta-actin, indicating that CUGBP2 may play an important role in the diminished apoB mRNA editing seen in the colon.

Coordinate alteration of multiple enzyme components is associated with different levels of apoB mRNA editing in different tissues and cell lines. ApoB mRNA editing varies significantly in different tissues. To further investigate the correlation between editing levels and gene expression of the editing enzyme components, several tissues and cell lines were analyzed. As shown in Fig. 6, A and B, the editing for the mouse small intestine and liver and the mouse hepatocellular cell line BNL CL2 was 94%, 66%, and 5.2%, respectively. Compared with the small intestine, the mouse liver had increased levels of CUGBP2 (7.5-fold), GRY-RBP (1.9-fold), BAG4 (3.6-fold), ABBP1 (1.8-fold), and KSRP (6-fold). Compared with the small intestine, the hepatocellular cell line BNL CL2 had a dramatically decreased expression of ACF to an undetectable level by RT-PCR up to 40 PCR cycles (Fig. 6B and data not shown) and a dramatically increased CUGBP2 expression (over 30-fold). BNL CL2 cells also had decreased levels of apobec-1 (50%) and increased levels of GRY-RBP (12.5-fold), hnRNP-C1 (4.2-fold), BAG4 (4.8-fold), ABBP1 (12.5-fold), ABBP2 (2.9-fold), and KSRP (13.8-fold). These data indicate that the diminished editing in the liver and BNL CL2 cells compared with the small intestine is correlated with the significantly altered expression of multiple editing components.


Figure 6
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  		Fig. 6. Comparison of apoB mRNA editing and complex cofactor gene expression among tissues and cell lines. Total RNA was isolated from the mouse and rat adult small intestine and liver and FAO and BNL CL2 cells. cDNA was generated from the total RNA extract by the random primer method. cDNA aliquots were analyzed for apoB mRNA editing (A and C) or mRNA expression of multiple genes (B and D) by PCR amplification in the presence of [{alpha}-32P]dCTP. The resultant PCR products were resolved by 6% PAGE, and the gene expression levels were determined by a PhosphoImager.

 
When the rat small intestine and liver were compared, there was a relatively small difference in editing levels (84% vs. 70%, respectively) and there were no significant relative differences in component expression (Fig. 6, C and D). Compared with the rat small intestine, the rat hepatocellular cell line FAO had decreased editing (8.8%) associated with increased expression of GRY-RBP (3.2-fold), hnRNP-C1 (2.2-fold), BAG4 (3.1-fold), ABBP1 (2.8-fold), ABBP2 (2.1-fold), and KSRP (4.1-fold), whereas the expression of apobec-1 and ACF was similar. Interestingly, FAO cells had undetectable CUGBP2 levels even using PCR cycles as high as 40 while preserving the specificity of apoB mRNA editing as demonstrated by the absence of additional bands above apoB-48 in the editing primer extension assay, which are indicative of hyperediting (35) (Fig. 6 and unpublished data). These data further suggest that alteration of multiple enzyme components is associated with different levels of apoB mRNA editing in different tissues and cell lines.

All editing enzyme component candidates tested affect apoB mRNA editing as demonstrated by siRNA-mediated gene knockdown. All auxiliary editing components except apobec-1 and ACF have been proposed as candidate components of the editing complex based on their interaction or association with apobec-1, ACF, or apoB RNA. However, the exact composition of the editing complex remains largely uncertain. Compared with the small intestine, the diminished editing in cell lines BNL CL2 and FAO is associated with significant increases of the auxiliary editing components as described above. To further evaluate their roles and relationship with editing, individual gene knockdown experiments were performed in Caco-2 cells by siRNA methodology. Chemically synthesized annealed siRNA was transfected into Caco-2 cells, and, 24 h later, these Caco-2 cells were transfected with apobec-1-adenovirus. The siRNA-induced effects on editing and gene knockdown were analyzed with total RNA extracts as described in MATERIALS AND METHODS. As shown in Fig. 7, siRNA-mediated decreased expression of ACF, CUGBP2, BAG4, ABBP2, GRY-RBP, hnRNP-C1, KSRP, and ABBP1 (see Fig. 7, C, D, and F) was associated with the editing modulation in all cases (see Fig. 7, A, B, and E). The siRNA and apobec-1 transfection efficiency in Caco-2 cells was ~40–50% based on fluorescently labeled siRNA uptake, and the adenovirus-mediated transduction in Caco-2 cells varied from 30% to 90% depending on the adenoviral quantity applied (data not shown). Since the editing assay was done based on whole cell populations instead of only known transfected cells, the actual editing changes in transfected cells should be much higher than the data presented. Nevertheless, knockdown of ACF resulted in a 33% decrease in editing as expected due to its known essential function in the complex. Knockdown of CUGBP2, GRY-RBP, and hnRNP-C1 resulted in 2.18-, 1.71-, and 1.67-fold editing increases, respectively, which was consistent with their reported inhibitory function. Surprisingly, knockdown of KSRP, ABBP1, BAG4, and ABBP2 resulted in 1.75-, 1.40-, 1.59-, and 1.50-fold editing increases, respectively, although no obvious gene expression changes were observed for them during developmental editing upregulation in the mouse small intestine, indicating that they all may play direct or indirect inhibitory roles in the editing process.


Figure 7
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  		Fig. 7. Effect of siRNA-mediated gene knockdown on apoB mRNA editing and editing component levels in Caco-2 cells. Chemically synthesized annealed siRNA against each gene were transfected into Caco-2 cells by siPROT NeoFX. Twenty-four hours later, cells were infected with apobec-1-adenovirus, and total RNA was extracted from the cells 72 h later. Aliquots of cDNA generated by the random primer method were analyzed for apoB mRNA editing (A and B) or mRNA expression of different genes (C and D) by PCR amplification in the presence of [{alpha}-32P]dCTP as in Fig. 3. D: GAPDH expression levels for the same samples in C. E and F: graphical representation of the editing and gene expression above. Data for gene expression are normalized to the housekeeping gene GAPDH as a relative percentage for reference. Each bar in E and F represents the mean and SD of the group with n = 3.

 
To further investigate the properties of the siRNA-mediated gene knockdown, double-gene knockdown experiments were evaluated. As shown in Fig. 8A, the decreased editing effect of ACF siRNA (51% of control) was rescued by CUGBP2 siRNA to near control levels (94% of control) and partially rescued by GRY-RBP (70%), hnRNP-C1 (62%), and ABBP2 siRNA (70%). The 1.7-fold increased editing effect of CUGBP2 siRNA was eliminated by ACF siRNA (Fig. 8B). No additional effect was observed with CUGBP2 siRNA together with other cofactors' siRNA. The 1.4-fold increased effect of GRY-RBP siRNA (Fig. 8C) was reversed by ACF siRNA (70% of control) and further increased by CUGBP2 siRNA (1.8-fold). Other cofactors' siRNA had no additional effect above that of GRY-RBP siRNA.


Figure 8
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  		Fig. 8. Effect of double-siRNA-mediated gene knockdown on apoB mRNA editing in Caco-2 cells. Single or double chemically synthesized annealed siRNA was transfected into Caco-2 cells by siPROT NeoFX as in Fig. 7. Twenty-four hours later, cells were infected with apobec-1-adenovirus, and total RNA was extracted from the cells 72 h later. Aliquots of cDNA generated by the random primer method were analyzed for apoB mRNA editing. A: editing effect of ACF siRNA and combination of ACF siRNA with others. Scrambled siRNA was used as a negative control. B: editing effect of CUGBP2 siRNA and combination of CUGBP2 siRNA with others. C: editing effect of GRY-RBP siRNA and combination of GRY-RBP siRNA with others. Each bar in the graphs represents the mean and SD of the group with n = 3.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Coordinated expression of editing components during apoB mRNA editing upregulation and the importance of CUGBP2. ApoB mRNA editing is an exquisitely precise nuclear process that alters a single nucleotide in a molecule containing over 14,000 bases, playing a central role in lipid metabolism. ApoB mRNA editing in vivo is under tissue-specific, developmental, and metabolic regulation without compromise on editing specificity. Although it has been investigated extensively, the exact molecular mechanisms that regulate apoB mRNA editing remain largely unknown because of limited information on the relatively large number of identified editing complex components. Most studies have been done with the rat liver and primarily report only alterations in apobec-1 and ACF. Among the different situations where apoB mRNA editing is altered, the developmental regulation in the rodent small intestine is of particular interest because editing increases so dramatically from ~1% to ~90% in a very short period (12, 30, 31, 39). In this study, we investigate the temporal changes of editing in multiple models by assessing the expression of the multiple editing enzyme components relative to the magnitude of editing and are the first to evaluate the contribution of these various editing enzyme components in determining editing activity in the complex.

We found that the modulation of multiple components is involved in regulating apoB mRNA editing during mouse small intestine development. The core enzyme components, apobec-1 and ACF, were upregulated contemporaneously with editing. In contrast, the inhibitory enzyme components, CUGBP2, GRY-RBP, and hnRNP-C1, were downregulated in patterns that were temporally different than editing. The downregulation of CUGBP2 occurred 1 day before the major editing increase, whereas GRY-RBP and hnRNP-C1 were downregulated primarily 1–2 days afterward. Meanwhile, there were no significant or consistent changes in the expression of KSRP, ABBP1, ABBP2, and BAG4. These data are consistent with the known activities of these components. Increasing the expression of the active components, apobec-1 and ACF, while decreasing the expression of inhibitory components, CUGBP2, GRY-RBP, and hnRNP-C1, allowed the dramatic upregulation of apoB mRNA editing observed in the small intestine. The temporal changes of these five components suggest that there is a coordinated modulation in vivo. The dramatic 89% decrease of CUGBP2 expression prior to the upregulation of editing suggests that it may play an especially important physiological role as an initial responding component.

The human intestinal cell line Caco-2 is an in vitro cellular model for the developmental induction of apoB mRNA editing (15, 16). The increased editing observed in Caco-2 cells during culture on filters was associated with the altered expression of mainly apobec-1 and CUGBP2 and temporally corresponded with the increased apobec-1 expression, indicating that the active component apobec-1 plays a critical determinant role. The modulation of CUGBP2 expression represented the major difference between Caco-2 cells and the small intestine. CUGBP2 expression in Caco-2 cells cultured on either filters or plastic increased about 3-fold prior to the major editing changes, whereas CUGBP2 decreased 89% in the mouse small intestine. Consistent with this finding, CUGBP2 expression was also 2.7-fold relatively higher in the large intestine than the small intestine in association with the respective editing differences of 84% and 48%. CUGBP2 is a potent inhibitory component of the editing enzyme complex (3). The increased expression of CUGBP2 thus provides a basis for the lower level of apoB mRNA editing observed in Caco-2 cells and the large intestine and further indicates the important contribution of CUGBP2 in editing regulation.

The functional role of the editing enzyme components in apoB mRNA editing was evaluated in this study by assessing their mRNA expression levels relative to the magnitude of editing. It could be more informative to see a correlation between protein levels and editing activity since mRNA levels do not always correlate with protein levels. However, the cellular localization of protein affects editing activity because apoB mRNA editing is a nuclear event occurring temporally and perhaps physically in proximity to splicing and polyadenylation. The editing component protein will only be functional when it is transported to the correct nuclear position so that it can interact with other factors (6, 11). Because of these facts, even protein level determinations using whole cells still may have cause-effect interpretation limitations. Moreover, this optimal approach is limited by the sensitivity of protein assays and the small amount tissues available from the mouse fetal small intestine during development. mRNA expression values have shown their usefulness in a broad range of applications because mRNA has a key position as an intermediate in the flow of genetic information and has a much higher sensitivity of detection by RT-PCR. Many of the efforts at correlating mRNA and protein expression have been conducted in yeast. Overall, a significant relationship between mRNA and protein levels was observed in the global analysis of protein expression in yeast, although individual genes with equivalent mRNA levels can result in large differences in protein abundance (13, 17, 18). In addition, the conclusion in this study is based on mRNA expression patterns composed of a series of mRNA level alterations at multiple time points to minimize potential bias.

Absence of certain editing components in widely used cell lines. Many apoB mRNA editing studies have been performed on widely available cell lines such as Caco-2, Hep G2, FAO, McA7777, and BNL CL2. When compared with liver or small intestine tissues, the gene expression of the editing components are significantly different for these cell lines. Some components are even missing in Hep G2, FAO, McA7777, and BNL CL2 cells, whereas Caco-2 has all of those components. CUGBP2 expression was undetectable in the rat liver-derived cell line FAO (see Fig. 6). CUGBP2 was also at an undetectable level in McA7777 cells and was barely detectable in Hep G2 cells but was abundant in Caco-2 and BNL CL2 cells (data not shown). The editing level in FAO cells was surprisingly low considering that apobec-1 and ACF levels were comparatively similar to the rat small intestine and CUGBP2 was absent (Fig. 6), indicating the probable role of other components such as increased GRY-RBP expression, another inhibitory cofactor. In the mouse liver-derived cell line BNL CL2, ACF expression was undetectable by RT-PCR up to 40 cycles (Fig. 6). It appears that ACF expression is either at a very low level or absent in BNL CL2 cells. Compared with the mouse small intestine, the expression of CUGBP2 and GRY-RBP was 34- and 12.5-fold higher, respectively, indicating that CUGBP2 and GRY-RBP were in excess relative to ACF expression. It has been reported that CUGBP2 exerts its inhibitory effects by inhibiting the interaction between apobec-1 and ACF, whereas GRY-RBP exerts its inhibitory effects through binding to and sequestering ACF in the in vitro editing reaction (3, 7). The combination of very low ACF expression and high expression of ACF counteracting CUGBP2 and GRY-RBP suggests that additional factor(s) other than ACF complement apobec-1 to observe ~5% editing in BNL CL2 cells. ACF may not be the only required complement factor. These data also indicate that caution should be taken when interpreting data based on these cell lines.

Inhibitory effect of multiple editing components. Compared with the gene expression pattern of the editing components in the small intestine, cell lines including Caco-2, Hep G2, McA7777, FAO and BNL CL2 all have significantly increased expression of CUGBP2, GRY-RBP, hnRNP-C1, KSRP, ABBP1, ABBP2, and BAG4 (Fig. 6 and data not shown). siRNA-mediated gene-specific knockdown of these genes in Caco-2 cells all resulted in editing increases (see Fig. 7), suggesting that they all have a direct or indirect inhibitory effects on editing, although some of them did not have significant gene expression changes during the developmental upregulation of apoB mRNA editing. This inhibitory effect of these genes is consistent with the correlation between high expression of these genes and low editing levels in these cell lines. The Caco-2 cell line was chosen for the siRNA experiments because it was the only cell line possessing all of the candidate editing components.

Caco-2 cells have little editing ability before reaching 100% confluency and then differentiating due to very low apobec-1 expression. The effect of each individual gene knockdown by siRNA on editing in Caco-2 cells was detected by overexpression of apobec-1 with adenovirus after the transfection of siRNA, and the editing levels were determined on whole cell population. This Caco-2 system provides reproducible results in different sets of experiments. Similar patterns of the siRNA effect were also obtained with human liver cell line H3B with low magnitude. The siRNA effect in HepG2 cells was complicated because of toxicity to adenovirus. The siRNA gene knockdown in Caco-2 cells by siPORT NeoFX, which mediated transfection while cells were plated, was not as low as desired, probably reflecting a relatively low transfection efficiency of siRNA (40–50%, data not shown). This was the best method we found and consistent effects upon editing were obtained. The actual editing levels represent a low estimation since only a fraction of cells were transfected with both siRNA and apobec-1 and the quantitation was based on the entire cellular mRNA.

Previously published gene knockdown reports of apoB mRNA editing components have only utilized antisense oligonucleotide or antisense cDNA expression methods. Antisense oligonucleotide-mediated gene knockdown in McA7777 cells resulted in an editing decrease when targeted against apobec-1 and ACF and an editing increase when targeted against CUGBP2 and GRY-RBP (3). Antisense cDNA expression-mediated knockdown by transient transfection of the antisense cDNA in pHOOK3 vector in apobec-1-overexpressed BNL CL2 and/or McA7777 cells followed by selection of tranfected cells showed that there was decreased editing with ACF, ABBP1, and ABBP2; increased editing with BAG4; and no effect with GRY-RBP (21, 24, 25). Our siRNA-mediated gene knockdown report demonstrated an editing decrease with siRNA against ACF and increase with those against CUGBP2, GRY-RBP, hnRNP-C1, KSRP, ABBP1, ABBP2, and BAG4. The results on ACF, CUGBP2, GRY-RBP, and BAG4 are consistent with previous reports. The result on hnRNP-C1 is also consistent with its in vitro inhibitory property, as previously reported (19). The inhibitory effect of KSRP is a new finding. However, our results on ABBP1 and ABBP2 are opposite to those obtained using the antisense cDNA expression method. Although differences in cell lines or experimental conditions may contribute to these different results, another reason could be nonspecific effects induced by antisense cDNA expression. Double-strand RNA may be formed between expressed antisense cDNA and target mRNA and, when in the presence of ribonuclease Dicer, could generate many nonspecific siRNA (29). The functions of ABBP1 and ABBP2 need to be further clarified.

In summary, the simultaneous significant modulation of apobec-1, ACF, CUGBP2, GRY-RBP, and hnRNP-C1 during development in the mouse small intestine demonstrates that a coordinate expression of multiple editing components is involved in the upregulation of editing. Besides apobec-1 and ACF, CUGBP2 may also play an important role since it is the first modulated factor during editing upregulation in the mouse small intestine and Caco-2 models. The preservation of editing specificity during the dramatic developmental upregulation of editing and the known loss of specificity observed when apobec-1 is overexpressed indicates that the appropriate relative expression of editing component is necessary for editing specificity. In conclusion, a coordinated expression of various apobec-1 components preserves the specificity and determines the magnitude of apoB mRNA editing.


   ACKNOWLEDGMENTS
 
We thank Dr. Xiaobin Zhong for the instruction and help with the animal studies.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. P. Patterson, Office of Biotechnology Activities, National Institutes of Health, 6705 Rockledge Dr., Suite 750, Bethesda, MD 20892 (e-mail: pattersa{at}od.nih.gov)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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